Exploring the role of ATP1A3 mutations in sudden unexplained death in epilepsy

项目总结/摘要 癫痫不明原因猝死（SUDEP）是指有癫痫发作史的患者突然不明原因死亡 和癫痫病患者的健康状况。它是癫痫患者死亡的主要原因， 神经系统死亡的原因被认为是由心脏和神经因素的顶点引起的癫痫， 诱发的心动过缓是SUDEP的危险因素，并可能在易感心肌中触发致死性室性心律失常。 开发一个概括这一点的强大实验模型将为基础研究打开大门， 开发关键的药物疗法。SUDEP是儿童交替性偏瘫（AHC）患者的悲惨结局 其特征在于癫痫、肌张力障碍、瘫痪，以及特别是在心动过缓的情况下的猝死。之间 在AHC患者中，90%的患者在ATP 1A 3编码的α-3催化亚基中存在潜在的病理性遗传变异， Na/K ATP酶泵（ATP 1A 3）。我们已经确定了最常见的AHC相关性之间的强相关性， ATP 1A 3变异体（D801 N）和心动过缓时心电图短QT和室颤。人诱导多能 来自ATP 1A 3-D801 N阳性儿童（hiPSC-CMD 801 N）干细胞衍生的心肌细胞显示缩短 复极时间、钙稳态破坏和后去极化延迟，这些都是心律失常的触发因素。 携带D801 N变异体（Atp 1a 3D 801 N）的敲入小鼠具有癫痫发作、心动过缓和猝死。此外，Atp 1a 3D 801 N 与对照组相比，小鼠具有室性心律失常的倾向，特别是在较低的心率下。总的来说， 这些发现提出了心脏中ATP 1A 3的破坏是AHC患者SUDEP的基础的可能性， 心动过缓引发的心律失常我们假设D801 N降低泵功能，导致复极缩短 并容易导致致命的室性心律失常我们的目标是使用AHC作为模型来确定ATP 1A 3在其中的角色 探讨SUDEP的发生机制，并探讨心动过缓的预防作用。为了解决这个问题， 一种创新和严格的方法，我们将利用患者来源的hiPSCD 801 N-和小鼠Atp 1a 3D 801 N为基础的模型， 体外、离体和体内研究，以确定心脏中心律失常易感性的机制。我们特别 拟确定1）D801 N诱导心肌细胞动作电位时程缩短的机制，2） 在3D组织模型和离体分析中由D801 N诱导的心脏血管生成的机制，以及3）是否 癫痫发作引起的心动过缓是Atp 1a 3D 801 N小鼠“脆弱”心肌的致瘤性触发因素。在 为了实现这些目标和总体目标，我们将确定ATP 1A 3在心血管生理学中的功能， 在心脏复极、钙信号传导和室性心律失常中的作用，从而确定了 药物治疗.最后，这将建立强大和严格的模型，以确定猝死的机制， AHC将提供更广泛的SUDEP机制的见解。

PROJECT SUMMARY/ABSTRACT Sudden unexplained death in epilepsy (SUDEP) is the sudden and unexplained death of a patient with a history of seizures and epilepsy who is in a reasonable state of health. It is a major cause of death in patients with epilepsy and a common cause of neurologic death overall. Believed to be caused by a culmination of cardiac and neurologic factors, epilepsy induced bradycardia is a risk factor for SUDEP and may trigger lethal ventricular arrhythmias in susceptible myocardium. Development of a robust experimental model which recapitulates this would open the door for foundational studies to develop critical pharmacotherapies. SUDEP is a tragic outcome in patients with alternating hemiplegia of childhood (AHC) which is characterized by epilepsy, dystonia, paralysis, and, notably, sudden death in the setting of bradycardia. Among AHC patients, 90% harbor underlying pathologic genetic variants in the ATP1A3-encoded alpha-3 catalytic subunit of the Na/K ATPase pump (ATP1A3). We have identified a strong correlation between the most common AHC-associated ATP1A3 variant (D801N) and short QT on ECG and ventricular fibrillation during bradycardia. Human induced pluripotent stem cell-derived cardiac myocytes from an ATP1A3-D801N-positive child (hiPSC-CMD801N) demonstrate shortened repolarization time, disrupted calcium homeostasis, and delayed-after depolarizations, which are triggers for arrhythmias. Knock-in mice hosting the D801N variant (Atp1a3D801N) have seizures, bradycardia, and sudden death. Further, Atp1a3D801N mice have a predisposition to ventricular arrhythmias, particularly at lower heart rates, compared to controls. Collectively, these findings raise the possibility that disruption of ATP1A3 in the heart underlies SUDEP in AHC patients through bradycardia-triggered arrhythmias. We hypothesize that D801N reduces pump function leading to shortened repolarization time and predisposes to lethal ventricular arrhythmias. Our goal is to use AHC as a model to establish the role ATP1A3 in the heart, determine the mechanism of SUDEP, and explore the proarrhythmic effect of bradycardia. To approach this in an innovative and rigorous way, we will utilize patient-derived hiPSCD801N- and murine Atp1a3D801N-based models for in vitro, ex vivo, and in vivo studies to determine the mechanism of arrhythmia predisposition in the heart. Specifically, we propose to determine 1) the mechanism of action potential duration shortening induced by D801N in cardiac myocytes, 2) the mechanism of cardiac arrhythmogenesis induced by D801N in 3D tissue models and ex vivo analysis, and 3) whether bradycardia due to seizures is an arrhythmogenic trigger for a “vulnerable” myocardium in Atp1a3D801N mice. In accomplishing these aims and overall goal, we will determine the function of ATP1A3 in cardiovascular physiology and its role in cardiac repolarization, calcium signaling, and ventricular arrhythmias, thus identifying molecular targets for pharmacotherapy. Finally, this will develop robust and rigorous models to determine the mechanisms of sudden death in AHC and will provide insights into mechanisms of SUDEP more broadly.